Abstract Objectives RF and ACPA are used as diagnostic tools and their presence has been associated with clinical response to some biologic DMARDs (bDMARDs) in RA. This study compared the impact of seropositivity on drug discontinuation and effectiveness of bDMARDs in patients with RA, using head-to-head comparisons in a real-world setting. Methods We conducted a pooled analysis of 16 observational RA registries. Inclusion criteria were a diagnosis of RA, initiation of treatment with rituximab (RTX), abatacept (ABA), tocilizumab (TCZ) or TNF inhibitors (TNFis) and available information on RF and/or ACPA status. Drug discontinuation was analysed using Cox regression, including drug, seropositivity, their interaction, adjusting for concomitant and past treatments and patient and disease characteristics and accounting for country and calendar year of bDMARD initiation. Effectiveness was analysed using the Clinical Disease Activity Index evolution over time. Results Among the 27 583 eligible patients, the association of seropositivity with drug discontinuation differed across bDMARDs (P for interaction <0.001). The adjusted hazard ratios for seropositive compared with seronegative patients were 1.01 (95% CI 0.95, 1.07) for TNFis, 0.89 (0.78, 1.02)] for TCZ, 0.80 (0.72, 0.88) for ABA and 0.70 (0.59, 0.84) for RTX. Adjusted differences in remission and low disease activity rates between seropositive and seronegative patients followed the same pattern, with no difference in TNFis, a small difference in TCZ, a larger difference in ABA and the largest difference in RTX (Lundex remission difference +5.9%, low disease activity difference +11.6%). Conclusion Seropositivity was associated with increased effectiveness of non-TNFi bDMARDs, especially RTX and ABA, but not TNFis.